Cell Culture

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Why are my cells not attaching or proliferating after thawing?

20 min Read

Thawing a pizza or frozen desert to snack on might be second nature to many of us, but in the world of cell culture, thawing cells from Ultra-low Temperature Freezers or Cryogenic Liquid Nitrogen Storage Tanks can be a tricky, delicate process. Unlike frozen pizzas, we can’t just put frozen cells into a preheated oven to warm them up. Multiple things can go wrong in the thawing process that can end up affecting the viability of the cells. In this article, we will be diving into why some cells refuse to attach or proliferate after being thawed out and offer some tips to avoid this happening to you!

Cell Culture Thawing Protocol

Cell Thawing: Best Practices

Waterbaths are an important tool for thawing - here are some important factors to take into account during the thawing procedure, which should take at most 10 minutes to ensure that the cells are happy:

a. Thaw cells quickly. Place the lower half of the vial into a 37°C water bath while agitating gently. Remove after 60 seconds. There should still be a few ice crystals left after thawing.

Thawing cells quickly prevents recrystallization both outside and inside the cells, as small crystals may re-form if thawing is too slow, causing more ice injury.

b. Do not over-thaw. DMSO is toxic at room temperature so it is recommended to thaw your cells just until there is the tiniest ice crystal in the cryovial.

If left at room temperature for an hour with exposure to 5% DMSO, cell viability could decrease by as much as 10%. This is due to the DMSO interacting with hydrophobic proteins in the cell, causing them to denature and become toxic to the cells.

c. Use pre-warmed media. Moving from a sub-zero environment straight to room temperature will put cells under a lot of stress. It's important to make sure that your cells are put into pre-warmed (either room temperature or 37°C) media right after rapid thawing as the media will help dilute the toxic DMSO as well.

d. Handle cells gently. Do not vortex or centrifuge cells at high speeds.

In order to reduce the possibility of further cell damage, centrifuging at high speeds is not recommended (around 1,500 rpm for 5 minutes will do the trick).

e. Avoid contamination. Keep the cryovial cap out of the waterbath’s water to avoid contamination.

Contamination is always a serious problem with cell culture work, so to minimize the potential of those pesky impurities and contaminants getting to our cells, you’ll need to make sure to keep the cryovial caps out of the waterbath’s water. You don’t know what kind of contaminants are swimming in there!

Cell Culture Thawing Protocol: Best Practices

Cell Thawing: Optimal Culturing Conditions

Now that your cells have been successfully thawed, the next step is to make sure that they have a suitable environment to grow in. Ensure the media and incubation conditions (temperature and CO2 levels) are set for optimal growth of your cells - this information will be provided by the manufacturer. Some cells, like the HEK293 and HeLa cells, are easy to maintain and can proliferate even if the conditions are not perfect, but some cells are more sensitive to small changes in the culturing conditions. It is important to keep the following two additional factors in mind when it comes to preparing culturing conditions:

a. Do not use heat-inactivated FBS unless specified (heat-inactivation of FBS is an old practice).

Heat-inactivating FBS involves heating the FBS serum to 56°C in a water bath for 30-40 minutes, which inactivates any chemicals or mycoplasma that could hinder cell growth. However, most of the FBS manufactured nowadays are filtered prior to packaging, removing the majority of mycoplasma potentially living in the medium.

Heat-inactivating the FBS has also been shown to inactivate complement proteins, and can affect other factors like vitamins and amino acids (among others) that are present in the serum. This can compromise the proliferation of your cells.

Some studies have also shown that heat-inactivated FBS will negatively affect the attachment of cells to cultureware, and increase the sensitivity of cells, causing their proliferation rates to decrease.

Aim for the optimal seeding density (cells/cm2) for each cell line.

When seeding density is too low (cells are too dilute): Cell-to-cell contact helps stimulate a variety of membrane-bound receptors involved in intercellular communication, stimulating the cells to secrete beneficial factors into the medium to condition it for themselves. If the seeding density is too low, it won’t be possible for them to create the optimal conditions. This will cause the cells to take much longer to proliferate, leading to detachment and more sensitive cells, which ultimately leads to faster senescence.

When seeding density is too high: Cells can also become overcrowded and stressed if seeding density is too high, resulting in having less room for growth, insufficient nutrients, and overaccumulation of metabolic waste in the medium. Frequent media changes will be required to prevent cell death from waste build up.

Additional factors:

  • Determining the optimal seeding density involves counting your live cells - to get an accurate count, make sure you only count the live cells.
  • The amount of media and ideal seeding densities needed are dependent on the flask/plate size that is used. Make sure you are adding the right amount of cells and media according to the handling information provided by the manufacturer.
  • Understanding the morphology and type of cells you are working with is also important, as some cells we encounter may be suspension cells. Those cells will not attach at all compared to adherent cells, so you don’t need to panic over your suspension cells not attaching whatsoever.

Cell Culture Thawing Protocol:Optimal Culturing Conditions

Attachment factors - is Extracellular Matrix (ECM) required?

Some cells are naturally slower to attach to culture surfaces, which means that they may also require an Extracellular Matrix (ECM). Extracellular Matrix is essential for cell communications and provides scaffolding for cells to help with attachment. Coating the vessels with ECM has been shown to mimic in vivo cell behavior, as ECM can store both growth and attachment factors.

If there are floating cells 24 hours after initial seeding, do not remove them:

  • Before removal, verify that the cells are non-viable using trypan blue or another viability assay. Alternatively, you can look under a microscope and see if the cells are “bright and shiny,” indicating they are alive and healthy.
  • Keep floating cells in culture until the first subculture. Removal of too many viable floating cells can result in a low cell density which triggers cells to enter the lag phase of growth. This can greatly increase time needed between subculturing and even cause the culture to collapse.

Read our Cell Culture: Viability and Cell Attachment article to learn how to recognize healthy or dead/compromised cell cultures.
Media and media components

Every cell line requires different media and media components for optimal proliferation and attachment - this information will be provided by the manufacturer. Here are some general tips to ensure you prepare and handle media correctly:

  • Glutamine is an essential amino acid for the production of different types of proteins that support the cell’s high energy demands. Glutamine degrades rapidly to toxic ammonia at 37°C in incubators, so opened bottles of media containing glutamine should be discarded after 4-6 weeks, or be re-supplemented with glutamine.
  • Aliquot your pre-warmed medium before use and use only the volumes you need for the day. This reduces how many times you need to touch the bottle’s lid and potentially introduce contaminants to the media.
  • Due to the increased sensitivity of cells following thawing, they may become more permeable to antibiotics. If antibiotic selection is needed, it is best to wait until the cells have recovered from thawing stress before introducing the antibiotics.

Cell Culture Thawing Protocol:Optimal Culturing Conditions

With enough patience and practice, even the most fragile and sensitive cells will be happy and ready to proliferate after being thawed. Now that you understand the ins and outs of cell thawing, you’re ready to coax your cells out of their hibernation in the freezer safely!

Download our general guidelines for thawing cryopreserved cells.